Post-doctoral - Thurnau Research Fellow, Department of Biological Chemistry, University of Michigan, March-December 1986
COURSES FOR CHEMISTRY:
CHEM 634
RESEARCH INTEREST:
Complexes of nucleic acid oligomers with photosensitizers serve as models for cooperative targeting of nucleic acid sequences in biological systems, with the application to in vivo anti-sense and in vitro diagnostic technologies. Moreover, such systems offer opportunities for studying electron and energy transfer through molecular systems consisting of stacked pi-bonded units. We are particularly interested in a set of related cationic porphyrins which display a range of binding modes with respect to nucleic acids that depend on the structure of the porphyrin as well as the sequence and structure of the nucleic acid. These are porphyrins tetra substituted at the meso position by nitrogen-containing aromatic rings.
In previous work, we demonstrated highly specific and efficient photo-sensitized cleavage of DNA by the cationic porphyrins TMAP (tetrakis [4-trimethyl anilinium] porphine and T4MpyP (tetrakis [4-methylpyridinyl] porphine (1). TMAP is very specific for three-way junction (3WJ) structures, in which it appears to intercalate. We have determined the structure of a DNA 3WJ using two-and three-dimensional NMR spectroscopy (2,3) and shown that it consists of two continuously stacked helices. This appears to create an intercalation site larger than is available between the stacked basepairs of ordinary duplex DNA, into which this porphyrin is too bulky to intercalate. T4MpyP on the other hand appears to be able to intercalate at specific sequences in duplex DNA as well as in 3WJ. We are currently applying NMR to investigate these interactions in a detailed manner to determine three-dimensional structures of porphyrin/DNA complexes, where the DNA components consist of oligomers designed to form duplexes or 3WJ.
The two-dimensional NOESY NMR data we have acquired indicate clearly that T4MPyP intercalates specifically at the 5'-CG-3' site of an 8-mer duplex DNA. We first carried out complete assignment of the proton resonances of the free DNA. Spectra were acquired in H2O solvent to monitor exchangeable protons which give valuable information regarding hydrogen-bonding. We then titrated in the porphyrin and were able to monitor specific interactions between the assigned DNA base and sugar protons with specific porphyrin protons. These data clearly show that intercalation is occurring as certain DNA-DNA NOEs are gone and replaced by DNA-Porphyrin interactions.
Molecular modeling studies are underway employing the AMBER suite of programs, which is especially designed for use with nucleic acids. The NMR NOE data re-incorporated in the form of distance constraints between specific protons on the DNA and on the ligand. These constraints are calculated using complete relaxation matrix methods that take into account spin diffusion effects. Molecular models for porphyrins were constructed using bond distances and angles taken from crystallographically determined structures and atom-centered charges were obtained from ab initio calculations using the 3-12G* basis set. These parameters were used with the AMBER forcefield to carry out simulated annealing and energy minimization of structures, incorporating the NMR constraints. Simulations were carried out with explicit solvent and counterions using the Ewald method for the final structure.
Other projects in the laboratory: 1) structure determinations of DNA duplexes containing modified nucleotides such as the easily oxidized diamino-purine which substitutes for adenosine and 2-thio thymidine, which substitutes for thymidine. These compounds offer exciting possibilities for anti-sense drug and diagnostic applications. 2) NMR, CD and molecular modeling studies of peptides which nucleate biomineralization. 3) Solid-state NMR studies of recombinant spider silk fibroin..
